1. Field of the Invention
The present invention relates to the fields of PCR and HLA-typing. More specifically, the present invention discloses methods and systems for a tandem PCR process to amplify DNA or RNA within a raw biological specimen and subsequent HLA-typing thereof on an individual or population scale in a field or medical office environment.
2. Description of the Related Art
There is a new and rapidly growing understanding of the medical significance of HLA typing in current medicine. As an example Table 1 demonstrates the very large range of diagnostic and public health applications for HLA-typing.
TABLE IClinical Correlation with AlleleClinical Utility of HLA-B ScreeningHLA-B AlleleB*07Ebola. Protective (with B*14). Non-B*07 + B*14 are highly enriched infatal response to Ebola.those who surviveB*0801Susceptibility to HIV-2 infectionHIV-2 screen for AIDSB*11Chlamydia trachomatis. ProtectivePredictive of those who do not developagainst blindness fromblindnessB*14Ebola. Protective (along with B*07).B*07 and B*14 are highly enriched inNon-fatal response to Ebolathose who survive.B*1502Adverse Drug Response:FDA: All Chinese should be screenedcarbamazepine-induced Stevens-before Rx.Johnson in ChineseB*1503Poor AIDS prognosis after HIV-2HIV-2 screen for AIDSinfectionB*17Leukemia in children. ALL highB*17 and A*33 may combine to predictleukocyte counts at presentation.male relapse.B*27Ankylosing Spondylitis (reactiveSeverity of reactive arthritis stronglyarthritis): B*27 explains 50% of risk.correlated with B*27.B*27Reactive Arthritis: B*27 associatedClinical B*27 Review in Nature.with enhanced risk of all forms ofsevere Reactive Arthritis.B*27Crohn's Disease: HLA-B*27 appearsB*27 not associated with absoluteto convey a very high risk ofCrohn's risk, but with subsequentdeveloping axial inflammation ininflammation.Crohn's disease.B*27HIV-1: All B*27 allelesAlso seems to correlate with early workProtective, HIV-1 Elite Controller.on vaccines.B*3503HIV-1: B*3501 is protective for HIVAs for B*57, a subtle change in B*35progression. Other B*35 allelesallele has significant effects.show rapid HIV progression.B*39Diabetes: Type I diabetes geneticWhole genome scanning study inrisk explained by B*39 Plus HLA-Nature.DQB1 and HLA-DRB1.B*4402Cervical Cancer: EnhancedHLA explains enhanced genetic risk forsquamous cell cervical cancer RISKcervical cancer. B*4402 is the centralwith one or more of A*0201-theme.Cw*0501, DRB1*0401, orDQB1*0301.B*51Behcet's disease: AutoimmuneB*51 is strongly correlated withdisease of the vasculature.severity.B*57HCV: Protective effect.All B*57s correlated with spontaneousrecovery.B*5701Adverse drug response: AbacavirDescribed as “Gold Standard: in 2009sensitivity.for personalized medicine.B*5701Adverse drug response: AbacavirLarge scale Australian screening trialsensitivity.confirms high predictive power forADR.B*5701HIV-1: Protective effect HIV-1 EliteThe role of B*5701 in HIV risk onController. Highest geneticinfection is the gold standard in hostcorrelation. HLA-C may be aeffects in infectious disease.secondary player.B*5702HIV-1: Not Protective HIV-1 EliteB*5701 and B*5702 must be cleanlyControllerresolved at high resolution for HIVprogression and Abacvir ADRB*5801Adverse drug response: AllopurinolHighly predictive of adverse responsesensitivity in Chinese.to allopurinolType II HLA AllelesDRB1*0401Type 1 Diabetes (T1D)Personalized T1D riskDRB1*0401Multiple Sclerosis (MS)Personalized MS riskDRB1*0401Rheumatoid Arthritis (RA)Personalized RA riskDRB1*0402Type 1 Diabetes (T1D)Personalized T1D riskType 1 + Type II Allele CombinationsB*4402,High Risk for Cervical Cancer afterCervical Cancer screening testDRB1*1101HPV infectionDQB1*0301Type II allele combinationsDQA1*0501:T1DPersonalized T1D riskDQB1*0201DQA1*0501:Celiac DiseaseKey marker for celiac and otherDQB1*0201Autoimmune diseasesDQA1*0501:T1DPersonalized T1D riskDQB1*0201DQA1*0501:Celiac DiseaseKey marker for celiac and otherDQB1*0201Autoimmune diseasesDRB1*0301:Highest known inherited risk for T1DPossible clinical and public healthDQA1*0501:screening test for T1DDQB1*0201DRB1*04:Second Highest inherited risk forPossible clinical and public healthDQA1*03:T1Dscreening test for T1DDQB1*0302DRB1*13Predictive of resistance to AIDSPossible clinical and public healthDQB1*06upon HIV-1 infectionscreening test for AIDSDRB1*15Correlated with high risk of cervicalPossible cervical cancer screening testDQB1*0602cancer After HPV infectionImportant HLA allele combinations with Genes other than HLA(B*57, B*27) +HLA-B*57 or B*27 types, whichPossible clinical and public healthKIR3DS1present Bw4-80I epitopes, pairedscreening test for AIDSwith activating KIR type 3DS1, havehighest power to predict resistanceto AIDS, upon HIV-1 infectionHLA-Bw4 +Poor survival prognosis for multiplePossible diagnostic screening tool inKIR3DS1myelomacancer
However, at present, HLA typing literally requires the effort of an entire molecular genetics laboratory. Incoming blood specimens must first be purified by methods such as spin columns or magnetic beads, followed by quantitation of the purified DNA by methods such as PicoGreen fluorimetry or UV absorbance. The quantified DNA is then subjected to PCR amplification and, following PCR, is then analyzed by high throughput re-sequencing or, more recently, by multiplex hybridization analysis by beads or by microarrays. Thus, the resulting workflow requires the effort of a full molecular genetics laboratory, and at least one full day to compile the final HLA-typing data. The complexity of such a standard workflow also introduces major concerns related to chain-of-custody and the requirement for complex and costly LIMS systems and workflow standard operating procedures, to keep track of sample flow through the several processing and analysis workstations.
Efforts to streamline the process have included obviating DNA purification. Previous attempts to perform PCR amplification from unpurified blood have been problematic even with the availability of variants of the Taq polymerase used for standard PCR. The use of raw blood as a PCR substrate has not yielded consistent results due to the extreme sample-to-sample variation in the white cell complement of blood and possible sample-to-sample variation in the very large excess of blood solutes which can interfere with the underlying PCR reaction.
Thus, there is a recognized need in the art for low equipment and consumable cost, high-throughput methods of gene amplification and HLA typing. Specifically, the present invention is deficient in a hands-free or automated, real-time high-resolution method of HLA typing without a need for first externally purifying the DNA from a sample. The present invention fulfills this long-standing need and desire in the art.